Compositions comprising cannabinoids for treatment of nausea, vomiting, emesis, motion sickness or like conditions

ABSTRACT

Cannabinoids, in particular CBD and CBDA and their acid derivatives are provided for use as an active pharmaceutical substance in the treatment of nausea, vomiting, emesis, motion sickness. In particular extracts of  cannabis  plants are presented which are rich in these substances and suitable for pharmaceutical use.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/502,822, filed Mar. 2, 2005, which application is a national stagefiling under 35 U.S.C. §371 of PCT International applicationPCT/GB2003/000451, filed Feb. 3, 2003, which was published under PCTArticle 21(2) in English.

Functional vomiting is the forceful expulsion of gastric contentsproduced by involuntary contraction of the abdominal musculature. Thisoccurs when the gastric fundus and lower oesophageal sphincter arerelaxed. Functional vomiting may be accompanied by nausea (an unpleasantfeeling that vomiting is about to occur). Nausea is associated withaltered physiological activity, including gastric hypomotility, andincreased parasympathetic tone. Nausea may precede and accompanyvomiting. They represent the patient's awareness of afferent stimuli tothe medullary vomiting centre.

Physiological vomiting is a functional condition that occurs in responseto a number of factors affecting the vomiting centre. It may also betriggered by peripheral factors such as ingestion of toxins, disturbanceof the vestibular system, peritoneal inflammation and bowel obstruction.It may also occur in disorders of delayed gastric emptying as, forexample, in diabetes and idiopathic gastroparesis.

Psychogenic vomiting may be self-induced or may occur involuntarily insituations that are anxiety inducing, threatening or in some wayregarded as distasteful by the subject. It is also possible thatpsychological factors leading to vomiting are culturally determined (forexample, eating exotic food may be considered repulsive in the subject'sown cultural group). Vomiting may also express hostility as whenchildren vomit during a temper tantrum or in certain conversiondisorders.

Nausea and vomiting may also be induced by cytotoxic chemotherapy andradiotherapy. Post-operatively, patients may also vomit and experiencenausea, which may be attributable to the anaesthetic and analgesicagents frequently administered concurrently.

There are therefore peripheral and central mechanisms which are involvedin the expression of nausea and frank vomiting. Existing therapies areavailable for the treatment of these conditions but they havelimitations, and there is a need for alternative treatments,particularly where these can exert their effect through a centralnervous mechanism.

Investigation of a number of agents in a conscious animal model in whichmotion sickness is induced has confirmed that extracts of cannabis havean anti-emetic effect. Conventionally, the anti-emetic effect ofcannabis has been ascribed to delta⁹-tetrahydrocannabinol (Δ⁹-THC). Theuse of a whole animal, conscious model to explore this effect, and theavailability of cannabis extracts containing predominantly onecannabinoid or another have allowed for more detailed analysis of thecontribution of specific cannabinoids.

Surprisingly, it has been found that the anti-emetic effect, in a modelof motion sickness in Suncus murinus (the Asian musk (house) shrew), isgreatest in high cannabidiol (CBD) (and/or its acid form CBDA)containing extracts rather than in high tetrahydrocannabinol (THC)(and/or its acid form THCA) containing extracts (greater than 50% CBDmore preferably greater than 80% most preferably greater than 90%relative to other cannabinoids present).

It is hypothesised that the results will extend to the propyl variant ofCBD, namely CBDV and its acid form CBDVA.

More particularly, the results were noted in extracts in which thecannabinoids were predominantly in their acid form since the extractswere prepared by a methanolic extraction and had not been subjected to adecarboxylation step by, for example, heating. It is possible thereforethat the therapeutic effects noted are due to the acid form of thecannabinoids. If it is the acid form of the cannabinoid that isresponsible for the observed therapeutic effect this is particularlysurprising since the acid forms of cannabinoids have not hitherto beenknown to exhibit therapeutic effects.

According to a first aspect of the present invention there is provided acannabis extract, rich in CBD and/or CBDA and/or the propyl variantsCBDV and/or CBDVA, for use in the manufacture of a medicament for thetreatment of nausea, vomiting, emesis, motion sickness or likeconditions.

By rich is meant greater than 2% w/w of CBD and/or CBDA and/or thepropyl variants CBDV and/or CBDVA, more particularly greater than 5%,more preferably still greater than 7%.

According to another aspect of the present invention there is providedthe use of CBD and/or CBDV in the manufacture of a medicament for thetreatment of nausea, vomiting, emesis, motion sickness or likeconditions.

According to another aspect of the present invention there is provided acannabinoid acid for use as an active pharmaceutical substance.

According to yet another aspect of the present invention there isprovided CBDA or CBDVA for use as an active pharmaceutical substance.

Preferably the active pharmaceutical substance is present as amedicament for the treatment of nausea, vomiting, emesis, motionsickness or like conditions.

In one embodiment the CBD and/or CBDA and/or CBDV and/or CBDVA arepresent with other cannabinoids as a mixture derived from a plantextract (CMBE, cannabis based medicinal extract).

Plant extracts are preferred as, in addition to one or morecannabinoids, they will contain other chemical entities that may providea beneficial effect either in their own right or in combination with theone or more cannabinoids. Such other chemicals include, for example,volatile oils e.g. terpene or carotene rich volatiles. Known terpenespresent in the CMBE include C₁₀ terpenes, e.g. mycerene, and pinenes andC₁₅ terpenes e.g. caryophyllene.

Preferably the CBD or CBDV and/or the acids thereof are present with THCor THCV and/or the acids thereof.

Alternatively the CBD or CBDV and/or the acids thereof are substantiallyfree (less than 10%, more preferably less than 5% and most preferablyless than 2% relative to other cannabinoids present) from othercannabinoids.

In another embodiment the CBD or CBDV and/or the acids thereof aresynthetic.

The invention also extends to methods of treating nausea, vomiting,emesis, motion sickness or like conditions with CBD or CBDV and/or theacids thereof, either as the sole active ingredient or in mixtures asplant extracts.

Whilst the observation has been made on an extract administeredintraperitoneally, the skilled man will appreciate that a medicament canbe prepared for administration by any suitable means. These include, butare not limited to, solids, semi solids, e.g. gels, liquids, sprays,aerosols, inhalers, vapourisers, enemas, rectal suppositories and thelike. The route of administration need not be intraperitoneally butcould be oral, buccal, sublingual, or by any other suitable route e.g.the respiratory tract, nasal tract and distal rectum.

A “plant extract” is an extract from a plant material as defined in theGuidance for Industry Botanical Drug Products Draft Guidance, August2000, US Department of Health and Human Services, Food and DrugAdministration Centre for Drug Evaluation and Research.

“Plant material” is defined as a plant or plant part (e.g. bark, wood,leaves, stems, roots, flowers, fruits, seeds, berries or parts thereof)as well as exudates.

The term “Cannabis plant(s)” encompasses wild type Cannabis sativa andalso variants thereof, including cannabis chemovars which naturallycontain different amounts of the individual cannabinoids, Cannabissativa subspecies indica including the variants var. indica and var.kafiristanica, Cannabis indica and also plants which are the result ofgenetic crosses, self-crosses or hybrids thereof. The term “Cannabisplant material” is to be interpreted accordingly as encompassing plantmaterial derived from one or more cannabis plants. For the avoidance ofdoubt it is hereby stated that “cannabis plant material” includes driedcannabis biomass.

In the context of this application the terms “cannabis extract” or“extract from a cannabis plant”, which are used interchangeablyencompass “Botanical Drug Substances (BDS)” derived from cannabis plantmaterial. A Botanical Drug Substance is defined in the Guidance forIndustry Botanical Drug Products Draft Guidance, August 2000, USDepartment of Health and Human Services, Food and Drug AdministrationCentre for Drug Evaluation and Research as: “A drug substance derivedfrom one or more plants, algae, or macroscopic fungi. It is preparedfrom botanical raw materials by one or more of the following processes:pulverisation, decoction, expression, aqueous extraction, ethanolicextraction, or other similar processes.” A botanical drug substance doesnot include a highly purified or chemically modified substance derivedfrom natural sources. Thus, in the case of cannabis, “botanical drugsubstances” derived from cannabis plants do not include highly purified,Pharmacopoeial grade cannabinoids.

“Botanical drug substances” derived from cannabis plants include primaryextracts prepared by such processes as, for example, maceration,percolation, extraction with solvents such as C1 to C5 alcohols (e.g.ethanol), Norflurane (HFA134a), HFA227 and liquid carbon dioxide underpressure. The primary extract may be further purified for example bysupercritical or subcritical extraction, vaporisation andchromatography. When solvents such as those listed above are used, theresultant extract contains non-specific lipid-soluble material. This canbe removed by a variety of processes including “winterisation”, whichinvolves chilling to −20° C. followed by filtration to remove waxyballast, extraction with liquid carbon dioxide and by distillation.

Preferred “cannabis extracts” include those which are obtainable byusing any of the methods or processes specifically disclosed herein forpreparing extracts from cannabis plant material. The extracts arepreferably substantially free of waxes and other non-specific lipidsoluble material but preferably contain substantially all of thecannabinoids naturally present in the plant, most preferably insubstantially the same ratios in which they occur in the intact cannabisplant.

Botanical drug substances are formulated into “Botanical Drug Products”which are defined in the Guidance for Industry Botanical Drug ProductsDraft Guidance, August 2000, US Department of Health and Human Services,Food and Drug Administration Centre for Drug Evaluation and Research as:“A botanical product that is intended for use as a drug; a drug productthat is prepared from a botanical drug substance.”

“Cannabinoids” may be highly purified, Pharmacopoeial Grade substancesand may be obtained by purification from a natural source or viasynthetic means. The cannabinoids will include, but are not limited to,tetrahydrocannabinoids, their precursors, alkyl (particularly propyl)analogues, cannabidiols, their precursors, alkyl (particularly propyl)analogues, and cannabinol.

In preferred embodiments of the invention the formulations compriseextracts of one or more varieties of whole Cannabis plants, particularlyCannabis sativa, Cannabis indica or plants which are the result ofgenetic crosses, self-crosses or hybrids thereof. The precisecannabinoid content of any particular cannabis variety may bequalitatively and quantitatively determined using methods well known tothose skilled in the art, such as TLC or HPLC. Thus, one may chose aCannabis variety from which to prepare an extract which will produce thedesired ratio of CBD or CBDV to THC or THCV. Alternatively, extractsfrom two of more different varieties may be mixed or blended to producea material with the preferred cannabinoid ratio for formulating into apharmaceutical formulation.

The preparation of convenient ratios of CBD, CBDV, CBDA and CBDVAcontaining medicines is made possible by the cultivation of specificchemovars of cannabis. These chemovars (plants distinguished by thecannabinoids produced, rather than the morphological characteristics ofthe plant) can be bred by a variety of plant breeding techniques whichwill be familiar to a person skilled in the art. Suitable methods aregiven in Example 3. Propagation of the plants by cuttings for productionmaterial ensures that the genotype is fixed and that each crop of plantscontains the cannabinoids in substantially the same ratio.

Horticulturally, it is convenient to grow chemovars producing e.g. CBDand CBDV as the predominant cannabinoid from cuttings. This ensures thatthe genotype in each crop is identical and the qualitative formulation(the proportion of each cannabinoid in the biomass) is the same. Fromthese chemovars, extracts can be prepared by the similar method ofextraction. Convenient methods of preparing primary extracts includemaceration, percolation, extraction with solvents such as C1 to C5alcohols (ethanol), Norflurane (HFA134a), HFA227 and liquid carbondioxide under pressure. The primary extract may be further purified forexample by supercritical or subcritical extraction, vaporisation andchromatography. When solvents such as those listed above are used, theresultant extract contains non-specific lipid-soluble material. This canbe removed by a variety of processes including chilling to −20° C.followed by filtration to remove waxy ballast, extraction with liquidcarbon dioxide and by distillation. Preferred plant cultivation andextract preparation methods are shown in the Examples. The resultingextract is suitable for incorporation into pharmaceutical preparations.

A detailed examination of the pharmacological differences between CBDand THC has revealed significant differences in these compounds andconsequently the finding that CBD and/or its acid CBDA appear to beresponsible for the therapeutic effects noted was surprising. THC isbound with high avidity to CB1 and CB2 receptors in cerebral cortex andother sites; CBD is relatively inactive against CB1 receptors andappears to have non-cannabinoid receptor pharmacological actions in thecentral nervous system. Without prejudice to the teaching of theinvention, it is possible that the anti-emetic effect of CBD and/or itsacid CBDA is mediated via a non-cannabinergic mechanism.

Table 1 below illustrates some of the differences between thesecannabinoids.

TABLE 1 Effect THC THCV CBD CBDV Reference CB₁ (Brain receptors) ++ ±Pertwee et CB₂ (Peripheral + − al, 1998 receptors) CNS EffectsAnticonvulsant † −− ++ Carlini et al, 1973 Antimetrazol − − GW DataAnti-electroshock − ++ GW data Muscle Relaxant −− ++ Petro, 1980Antinociceptive ++ + GW data Catalepsy ++ ++ GW data Psychoactive ++ −GW data Antipsychotic − ++ Zuardi et al, 1991 Neuroprotective + ++Hampson antioxidant activity* A J et al, 1998 Antiemetic ++ − Sedation(reduced + + Zuardi et al, spontaneous activity) 1991 Appetitestimulation ++ Appetite suppression ++ Anxiolytic − ++ GW dataCardiovascular Effects Bradycardia − + Smiley et al, 1976 Tachycardia +− Hypertension § + − Hypotension § − + Adams et al, 1977Anti-inflammatory ± ± Brown, 1998 Immunomodulatory/anti- inflammatoryactivity Raw Paw Oedema Test − ++ GW data Cox 1 GW data Cox 2 GW dataTNF

 Antagonism + + ++ ++ Glaucoma ++ + *Effect is CB1 receptor independent.† THC is pro convulsant § THC has a biphasic effect on blood pressure;in naïve patients it may produce postural hypotension and it has alsobeen reported to produce hypertension on prolonged usage. GW InternalReport No 002/000159.

Whilst it is known that THC can be used to control nausea and vomitingpre-operatively the effect of other cannabinoids or combinations or theeffect of the acid forms present in, for example, plant extracts was nothitherto known.

The applicants studied the effect of other cannabinoids as cannabisextracts, and particularly extracts containing predominantly CBD or itsacid form CBDA in Suncus murinus and in which an emetic response can beinduced by a motion stimulus. Compounds which are effective in this testhave therapeutic benefit in the treatment of motion-induced nausea andvomiting, and also these conditions when induced by other pathways inthe peripheral and central nervous systems.

The applicant has determined that, for example, extracts in which thecontent of CBD and/or CBDA is 2-20% w/w, and the content of THC and orTHCA is 0.1-2% w/w are particularly beneficial.

The invention is further illustrated with reference to the accompanyingfigures and examples:

FIG. 1 is a TLC from the methanolic extract of the high CBD chemovar G5(M16). It shows significant CBD and CBDA peaks at around 6 and 7 minutesand lesser amounts of THC and THCA at around 10 and 18 minutes.

FIG. 2 is the TLC the methanolic extract of the high THC chemovar G2(M6). It shows significant THC and THCA peaks at around 10 and 18minutes along with lesser amounts of CBD and CBDA at around 6 and 7minutes.

The figures shown in FIGS. 1 and 2 are quantitative.

FIG. 3 is a thin layer chromatography plate showing the methanolicextracts, and by way of comparison BDS (decarboxylated and extracted bysub critical liquid CO₂). The results confirm that the methanolicextracts comprise a high proportion of the respective cannabinoids THCand CBD in what was later shown to be their acid forms.

FIG. 4 illustrates the results obtained for G5, the upper trace beingfor the BDS (decarboxylated and extracted by sub critical liquid CO₂)and the lower trace for the methanolic extract. The CBD peak at 35minutes and the THC peak at 38 minutes are marked.

FIG. 5 illustrates the results obtained for G2, the upper trace beingfor the BDS (decarboxylated and extracted by sub critical liquid CO₂)and the lower trace for the methanolic extract. The CBD peak at 35minutes and the THC peak at 37 minutes are marked.

EXAMPLE 1

Extracts of cannabis can be prepared by a number of solvent extractiontechniques, including the use of organic solvents alone or in admixturewith water and under sub critical or supercritical conditions.Cannabinoids may be present in cannabis biomass as free cannabinoids andas the corresponding acidic precursors. Conventional methods ofpreparation involve the total extraction of free cannabinoids andprecursors with solvents such as lower alkyl alcohols, particularlymethanol. In this example, total extracts of a high THC, and a highCBD-containing chemovar were made using methanol.

Biomass from each chemovar was separately extracted in a column withmethanol at room temperature, and the pooled percolate was collected.Solvent was removed by evaporation in a rotary evaporator at atemperature not exceeding 43° C. The resulting high THC and high CBDextracts were dispersed in 5% Polysorbate 80/normal saline and aPolysorbate/saline vehicle was used as control. The high THC extract(M6) contained more than 10% of THC and/or THCA and less than 1% of CBDand/or CBDA. The high CBD extract (M16) contained more than 7.3% of CBDand/or CBDA and less than 2% of THC and/or THCA.

A preliminary HPLC analysis of the methanolic extracts (and comparativedata for a botanical drug substance (BDS), when prepared by sub criticalCO₂ extraction after decarboxylation) is given in the table 2 below:

TABLE 2 Comparative Comparative Methanolic Decarboxylated Decarboxylatedextract from a CO₂ extract from CO₂ extract CBD rich THC rich from CBDrich chemovar chemovar chemovar Extract/Analyte % w/w % w/w % w/w THCN.D. 64.2%   2.9% THCA 1.4% N.D. N.D. CBD 6.1% 1.1% 70.2% CBDA 49.9% N.D. N.D CBN N.D. 1.0% N.D.

As would be expected, the acid forms of the cannabinoid predominate inthe methanolic extract of the non-decarboxylated herb.

For the methanolic extracts the % w/w of the principal cannabinoid (sumof acid+neutral forms) is lower than found in the equivalent BDS. Again,this is to be expected due to the lower selectivity of the methanol, ascompared to liquid CO₂. Thus, the methanolic extraction gives rise tothe extraction of more non-target material and a diluting of the activecontent of the extract.

The content of the methanolic extract was confirmed by TLC analysis andchromatogram traces for the high THC and high CBD extracts are shown inFIGS. 1 and 2. The traces in FIGS. 1 and 2 indicate the acid form of thecannabinoid to be the principal component observed, with smaller amountsof the corresponding neutral cannabinoid also being detected.

Samples of the methanolic extracts were run on a TLC plate along withsome cannabinoid markers, and by way of comparison botanic drugsubstance (BDS) (decarboxylated and extracted by sub critical liquidCO₂). The results are illustrated in FIG. 3.

Further analysis of the methanolic extracts, and by way of comparisonthe BDS (decarboxylated and extracted by sub critical liquid CO₂), usinggas chromatography indicates the presence of terpenes at around the 14to 18 minute mark. It should be noted that because G.C. is operated ataround 250° C. any acid form of the CBD and THC present in the extractswill be decarboxylated and appear on the trace as CBD or THC. Theresults are illustrated in FIGS. 4 and 5.

The most significant difference between the lower traces of FIGS. 4 and5 is the difference between the THC and CBD peaks.

EXAMPLE 2

Adult Suncus murinus (30-89 g bodyweight) of either sex were injectedwith either G2 (M6) or G5 (M16) at a range of doses from 1.0, 2.0 or 4.0mg/kg (or vehicle), intraperitoneally, 30 minutes prior to a motionstimulus (1 Hz 40 mm amplitude of shaking for 10 minutes). The animalswere observed for any overt behavioural change. A number of emeticepisodes and the latency of onset were recorded. Data were expressed asthe mean±s.e.m., group size n=6 and statistically analysed using ANOVA,followed by Bonferronni Dunnett's T test. The results are summarised intable 3 below.

TABLE 3 Effect of Cannabis Extracts given Intraperitoneally on Frequencyof Latency to First Emesis (means ± s.e.m.) Dose Emetic Episodes, mean ±s.e.m. Treatment mg/kg Latency (secs) P value Number P Value Vehicle103.0 + 25.3 12.4 + 2.4  control M16 1.0 206.8 ± 33.9 <0.05 6.3 ± 1.4<0.05 High CBD 2.0 250.5 ± 84.5 <0.05 6.6 ± 2.4 <0.05 chemovar 4.0Increase N5 Increase N5 M6 1.0 † NDDC NDDC High THC 2.0 NDDC NDDCchemovar 4.0* <100 >18 <0.05 † NDDC no detectable difference fromcontrol *Increase in vomiting, reduction in latency

The results for the high CBD producing chemovar extract (M16) shown inTable 3 are not unlike U-shaped dose response curves noted for someother pharmacological actions of cannabinoids.

The lack of effect of the high THC producing chemovar extract in thistest system is surprising. Indeed, at high doses there appears to be anincrease in vomiting and reduction in latency. It was confirmed thatneither M6 nor M16 had emetic activity in their own right. These datafurther emphasise the differences noted in the pharmacological effectsof THC and CBD, which have been investigated by the applicant.

EXAMPLE 3 Growing of Medicinal Cannabis

Plants are grown as clones from germinated seeds, under glass at atemperature of 25° C.±1.5° C. for 3 weeks in 24 hour daylight; thiskeeps the plants in a vegetative state. Flowering is induced by exposureto 12 hour day length for 8-9 weeks.

No artificial pesticides, herbicides, insecticides or fumigants areused. Plants are grown organically, with biological control of insectpests.

The essential steps in production from seed accession to dried MedicinalCannabis are summarised as follows:

EXAMPLE 4 Determination of Cannabinoid Content in Plants and ExtractsIdentity by TLC a) Materials and Methods

-   Equipment Application device capable of delivering an accurately    controlled volume of solution i.e. 1 μl capillary pipette or micro    litre syringe.-    TLC development tank with lid-    Hot air blower-    Silica gel G TLC plates (SIL N-HR/UV254), 200 μm layer with    fluorescent indicator on polyester support.-    Dipping tank for visualisation reagent.-   Mobile phase 80% petroleum ether 60:80/20% Diethyl ether.-   Visualisation reagent 0.1% w/v aqueous Fast Blue B (100 mg in 100 ml    de-ionised water). An optional method is to scan at UV 254 and 365    nm.

b) Sample Preparation

i) Herbal Raw Material

Approximately 200 mg of finely ground, dried cannabis is weighed into a10 ml volumetric flask. Make up to volume using methanol:chloroform(9:1) extraction solvent.

Extract by ultrasound for 15 minutes. Decant supernatant and usedirectly for chromatography.

ii) Herbal Drug Extract

Approximately 50 mg of extract is weighed into a 25 ml volumetric flask.Make up to volume using methanol solvent. Shake vigorously to dissolveand then use directly for chromatography.

c) Standards

0.1 mg/ml delta-9-THC in methanol.

0.1 mg/ml CBD in methanol.

The standard solutions are stored frozen at −20° C. between uses and areused for up to 12 months after initial preparation.

d) Test Solutions and Method

Apply to points separated by a minimum of 10 mm.

-   -   i) either 5 μl of herb extract or 1 μl of herbal extract        solution as appropriate,    -   ii) 10 μl of 0.1 mg/ml delta-9-THC in methanol standard        solution,    -   iii) 10 μl of 0.1 mg/ml CBD in methanol standard solution.

Elute the TLC plate through a distance of 8 cm, then remove the plate.Allow solvent to evaporate from the plate and then repeat the elutionfor a second time (double development).

The plate is briefly immersed in the Fast Blue B reagent until thecharacteristic red/orange colour of cannabinoids begins to develop. Theplate is removed and allowed to dry under ambient conditions in thedark.

A permanent record of the result is made either by reproduction of theimage by digital scanner (preferred option) or by noting spot positionsand colours on a tracing paper.

Assay THC, THCA, CBD, CBDA and CBN by HPLC

a) Materials and methods

-   Equipment: HP 1100 HPLC with diode array detector and autosampler.    The equipment is set up and operated in accordance with in-house    standard operating procedures (SOPlab037)

HPLC column Discovery C8 5 μm, 15 × 0.46 cm plus Kingsorb ODS2 precolumn5 μm 3 × 0.46 cm. Mobile Phase Acetonotrile:methanol:0.25% aqueousacetic acid (16:7:6 by volume) Column Operating 25° C. Temperature FlowRate 1.0 ml/min Injection Volume 10 μl Run time 25 mins DetectionNeutral and acid cannabinoids 220 nm (band width 16 nm) Referencewavelength 400 nm/bandwidth 16 nm Slit 4 nm Acid cannabinoids areroutinely monitored at 310 nm (band width 16 nm) for qualitativeconfirmatory and identification purposes only. Data capture HPChemistation with Version A7.01 software

b) Sample Preparation

Approximately 40 mg of Cannabis Based Medicinal Extract is dissolved in25 ml methanol and this solution is diluted to 1 to 10 in methanol. Thisdilution is used for chromatography.

0.5 ml of the fill solution, contained within the Pump Action SublingualSpray unit, is sampled by glass pipette. The solution is diluted into a25 ml flask and made to the mark with methanol.

200 μl of this solution is diluted with 800 μl of methanol.

Herb or resin samples are prepared by taking a 100 mg sample andtreating this with 5 or 10 ml of Methanol/Chloroform (9/1 w/v). Thedispersion is sonicated in a sealed tube for 10 minutes, allowed to cooland an aliquot is centrifuged and suitably diluted with methanol priorto chromatography.

c) Standards

External standardisation is used for this method. Dilution of stockstandards of THC, CBD and CBN in methanol or ethanol are made to givefinal working standards of approximately accurately 0.1 mg/ml. Theworking standards are stored at −20° C. and are used for up to 12 monthsafter initial preparation.

Injection of each standard is made in triplicate prior to the injectionof any test solution. At suitable intervals during the processing oftest solutions, repeat injections of standards are made. In the absenceof reliable CBDA and THCA standards, these compounds are analysed usingrespectively the CBD and THC standard response factors.

The elution order has been determined as CBD, CBDA, CBN, THC and THCA.Other cannabinoids are detected using this method and may be identifiedand determined as necessary.

d) Test Solutions

Diluted test solutions are made up in methanol and should containanalytes in the linear working range of 0.02-0.2 mg/ml.

e) Chromatography Acceptance Criteria:

The following acceptance criteria are applied to the results of eachsequence as they have been found to result in adequate resolution of allanalytes (including the two most closely eluting analytes CBD and CBDA)

i) Retention Time Windows for Each Analyte:

-   -   CBD 5.4-5.9 minutes    -   CBN 7.9-8.7 minutes    -   THC 9.6-10.6 minutes

ii) Peak Shape (Symmetry Factor According to BP Method)

-   -   CBD<1.30    -   CBN<1.25    -   THC<1.35

iii) A number of modifications to the standard method have beendeveloped to deal with those samples which contain late eluting impuritypeaks e.g method CBD2A extends the run time to 50 minutes. All solutionsshould be clarified by centrifugation before being transferred intoautosampler vials sealed with teflon faced septum seal and cap.

iv) The precolumn is critical to the quality of the chromatography andshould be changed when the back pressure rises above 71 bar and/oracceptance criteria regarding retention time and resolution, falloutside their specified limits.

f) Data Processing

Cannabinoids can be subdivided into neutral and acidic—the qualitativeidentification can be performed using the DAD dual wavelength mode.Acidic cannabinoids absorb strongly in the region of 220 nm-310 nm.Neutral cannabinoids only absorb strongly in the region of 220 nm.

Routinely, only the data recorded at 220 nm is used for quantitativeanalysis.

The DAD can also be set up to take UV spectral scans of each peak, whichcan then be stored in a spectral library and used for identificationpurposes.

Data processing for quantitation utilises batch processing software onthe Hewlett Packard Chemstation.

a) Sample Chromatograms

HPLC sample chromatograms for THC and CBD Herbal Drug extracts areprovided in the accompanying Figures.

EXAMPLE 5 Preparation of the Herbal Drug Extract

A flow chart showing the process of manufacture of extract from theHigh-THC and High-CBD (non acid and acid forms) chemovars is givenbelow:

EXAMPLE 6

High CBD cannabis was grown under glass at a mean temperature of 21+2°C., RH 50-60%. Herb was harvested and dried at ambient room temperatureat a RH of 40-45% in the dark. When dry, the leaf and flower head werestripped from stem and this dried biomass is referred to as “medicinalcannabis”.

Medicinal cannabis was reduced to a coarse powder (particles passingthrough a 3 mm mesh) and packed into the chamber of a SupercriticalFluid Extractor. Packing density was 0.3 and liquid carbon dioxide at apressure of 600 bar was passed through the mass at a temperature of 35°C. Supercritical extraction is carried out for 4 hours and the extractwas recovered by stepwise decompression into a collection vessel. Theresulting green-brown oily resinous extract is further purified. Whendissolved in ethanol BP (2 parts) and subjected to a temperature of −20°C. for 24 hours a deposit (consisting of fat-soluble, waxy material) wasthrown out of solution and was removed by filtration. Solvent wasremoved at low pressure in a rotary evaporator. The resulting extract isa soft extract which contains approximately 60% CBD with up to 4%tetrahydrocannabinol, within a total of other cannabinoids of 6%.Extracts were made using THCV and CBDV chemovars using the generalmethod described above.

A high THC chemovar was similarly treated and yielded an extractcontaining approximately 60% THC and approximately 6% of othercannabinoids of which 1-2% is cannabidiol and the remainder is minorcannabinoids including cannabinol. Quantitative yield was 9% w/w basedon weight of dry medicinal cannabis.

A person skilled in the art will appreciate that other combinations oftemperature and pressure (in the range+10° C. to 35° C. and 60-600 bar)can be used to prepare extracts under supercritical and subcriticalconditions.

EXAMPLE 7 Preparation of CBDA Summary of Process:

Yield:

100 g of G5 chemovar yields approx 5 g of purified CBDA.

Characteristics:

Pale yellow crystalline solid

Melting Point=45-48° C.

Chromatographic purity=94% CBDA by area normalisation

CBD 3%.

THCA non detected i.e. <0.1%

THC non detected i.e. <0.1%

Material slowly decarboxylates in solution

CBDA→CBD+CO₂

As CBDA does not co-elute with CBD during processing of the extract inthe low pressure column chromatography method employed, the detected CBDis likely to be formed from the breakdown of the CBDA during processingand analysis. This undesirable decarboxylation of the purified materialmight be minimised by manipulation of CBDA at sub-ambient temperatures.

EXAMPLE 8 Purification of CBD Overview of Process

Starting from freshly harvested plant material the process comprisesdrying and decarboxylation of the plant material, optional treatment(e.g. milling) of the dried plant material to reduce the particle size(preferably to less than 2000 μm), extraction with liquid carbondioxide, ethanolic precipitation to reduce the amount of non-targetmaterial, clean-up of the crude ethanolic extract by passage throughactivated charcoal, removal of solvent (ethanol) to produce aCBD-enriched fraction, and re-crystallisation of CBD from pentane.

Plant Material

GW Pharma Ltd has developed distinct varieties of Cannabis plant hybridsto maximise the output of the specific chemical constituents,cannabinoids. A “high CBD” chemovar designated G5 produces >90% totalcannabinoid content as CBD (naturally occurring in the plant in the formof CBDA). Alternative “high CBD” varieties can be obtained—see forexample, Common cannabinoids phenotypes in 350 stocks of cannabis, Smalland Beckstead, Lloydia vol 36b, 1973, p 144-156- and bred usingtechniques well known to the skilled man to maximise cannabinoidcontent.

Solvents

All solvents used in the isolation and analysis of CBD (e.g. n-pentane)were, unless otherwise stated, of chromatographic or A.R. grade.

Standards

Reference materials from Sigma were used as standards in the analysis ofextracts, intermediates and finished products, these were: Δ⁹ THC inmethanol BN 10601/B (ca. 1 mg/ml) and CBD in methanol BN 10601/C (ca. 1mg/ml).

Preparation of a Cannabidiol-Containing Extract

A cannabidiol-containing extract is prepared from a “high CBD” cannabischemovar according to the following process:

Prepare ethanolic solution of botanical drug substance as follows:

Extraction using liquid CO₂ is carried out under sub-critical conditionsat a temperature of approximately 10° C.±5° C. using a pressure ofapproximately 60 bar±10 bar. Decarboxylated plant material is packedinto a single column and exposed to liquid CO₂ under pressure forapproximately 8 hours, CO₂ mass flow 1250 kg/hr±20%.

Following depressurisation and venting off of the CO₂ the crude BDSextract is collected into sealed vessels. The crude BDS extract is heldat −20° C.±5° C.

The crude BDS extract contains waxes and long chain molecules. Removalis by “winterisation”, whereby the crude BDS extract is warmed to e.g.40° C.±4° C. to liquefy the material. Ethanol is added in the ratio of2:1 ethanol volume to weight of crude BDS extract. The ethanolicsolution is then cooled to −20° C.±5° C. and held at this temperaturefor approximately 48 hours.

On completion of the winterisation the precipitate is removed by coldfiltration through a 20 μm filter, to give an ethanolic solution of theBDS.

Preliminary charcoal clean-up may be carried out by passing theethanolic BDS solution (400-500 mg/ml) through a disposable plasticcolumn (130 mm×27 mm i.d.) packed with activated charcoal (decolourcarbDCL GDC grade, from Sutcliffe Speakman Carbons, 15.4 g per unit).Absolute ethanol B.P. (Hayman) is used as the solvent.

Ethanol and any water that may be present are removed by rotaryevaporation or thin film evaporation under reduced pressure (60° C.±2°C., with vapour at 40° C.±2° C./172 mbar and 72 mbar±4 mbar) to producea CBD-rich extract.

Solvent Re-Crystallisation

The CBD-rich extract is re-dissolving in a suitable solvent (e.g.n-pentane) and filtered to remove insoluble material. Solvent is thenremoved, e.g. by rotary evaporation, to produce crystalline CBD. Allsteps are carried out according to standard laboratory procedures, suchas would be known to those skilled in the art.

Product Characteristics Yield:

3 g of CBD BDS yields approx 1 g of purified CBD.

Characteristics:

White crystalline solid.

Chromatographic purity>99% CBD by area normalization.

Chromatographic purity superior to commercially available CBD Sigmastandard (refer to FIGS. 1 and 3).

THC non detected i.e. <0.1%

CBN non detected i.e. <0.1%

Identity confirmed by HPLC, GC and TLC retention behaviour compared toCBD Sigma standard.

Assay vs both Sigma CBD std in range 98.0-102.0%

Melting Point=64-66° C. (literature value=66-67° C.).

HPLC Analysis

The composition of the isolated products may be determined by HPLCanalysis.

A typical HPLC assay for

⁹ THC,

⁹ THCA, CBD, CBDA and CBN may be carried out as follows:

a) Materials and Methods Chromatography Equipment and Conditions:

Equipment Agilent (HP)1100 HPLC system with variable wavelength UVdetector or diode array detector. HPLC Column Discovery C8 5 μm 15 cm ×0.46 cm Pre-Column Kingsorb C18 5 μm 3 cm × 0.46 cm Mobile PhaseAcetonitrile:Methanol:0.25% w/v acetic acid (16:7:6 by volume) ColumnTemp 25° C. Flow Rate 1.0 ml min−1 Detection 220 nm 600 mA f.s.d. Secondwavelength 310 nm Injection Volume 10 μl Run Time 20-25 minutes (may beextended for samples containing small amount of late-eluting peaks)Elution Order CBD, CBDA, Δ⁹ THCV, CBN, Δ⁹ THC, CBC, Δ⁹ THCA

b) Sample Preparation

Samples of “pure” cannabidiol are diluted in methanol prior to HPLCanalysis. Optimal dilutions may be determined empirically.

Herbal cannabis samples are prepared by taking a 100 mg sample andtreating this with 5 or 10 ml of Methanol/Chloroform (9/1 w/v). Thedispersion is sonicated in a sealed tube for 10 minutes, allowed to cooland an aliquot is centrifuged and suitably diluted with methanol priorto chromatography.

c) Standards

Stock standard solutions of CBD, CBN and Δ⁹ THC in methanol atapproximately 1 mg ml⁻¹ are stored at −20° C. Diluted working standards(0.1 mg/ml for Δ⁹ THC and CBD and 0.01 mg/ml for CBN) are prepared inmethanol from the stock standards and stored at −20° C. (maximum periodof twelve months after initial preparation). After preparation, standardsolutions must be aliquoted into vials to reduce the amount of standardexposed to room temperature. Prior to use in an HPLC sample assay, therequired number of standard vials are removed and allowed to equilibrateto room temperature.

Injection of each standard is made in triplicate prior to the injectionof any test solution. At suitable intervals during the processing oftest solutions, repeat injections of standards are made. In the absenceof reliable CBDA and Δ⁹ THCA standards, these compounds are analysedusing respectively the CBD and Δ⁹ THC standard response factors.

d) Test Solutions

Diluted test solutions are made up in methanol and should containanalytes in the linear working range of 0.02-0.2 mg/ml.

e) Chromatography Acceptance Criteria:

The following acceptance criteria are applied to the results of eachsequence as they have been found to result in adequate resolution of allanalytes (including the two most closely eluting analytes CBD and CBDA)

TABLE 4 Retention time windows and Relative Retention Time (RRT) to Δ⁹THC for each analyte Retention time Cannabinoid (minutes) RRT (THC) CBD5.1-5.8 0.58 CBN 7.4-8.3 0.83 Δ⁹ THC  9.0-10.0 1.00 CBDA 5.5-6.2 0.615Δ⁹ THCV 5.9-6.2 0.645 CBC 11.6-12.8 1.30 Δ⁹ THCA 14.6-16.0 1.605

TABLE 5 Peak Shape (Symmetry Factor according to British Pharmacopoeiamethod) Cannabinoid Symmetry factor CBD <1.30 CBN <1.25 Δ⁹ THC <1.35

f) Data Processing

Cannabinoids can be subdivided into neutral and acidic—the qualitativeidentification can be performed using the DAD dual wavelength mode.Acidic cannabinoids absorb strongly in the region of 220 nm-310 nm.Neutral cannabinoids only absorb strongly in the region of 220 nm.

Routinely, only the data recorded at 220 nm is used for quantitativeanalysis.

The DAD can also be set up to take UV spectral scans of each peak, whichcan then be stored in a spectral library and used for identificationpurposes.

Data processing for quantitation utilises batch processing software onthe Hewlett Packard Chemstation.

g) Calculation:

Chromatographic purity of cannabinoid samples is calculated as a % oftotal cannabinoid content by area normalization.

Capillary Gas Chromatography (GC) Analysis a) Chromatography Equipmentand Conditions

Equipment Agilent (HP) 5890 or 6890 GLC system with HP7673 Autosamplerand FID detector GLC column SE54(EC5) 30 m × 0.32 mm i.d. (Alltech)phase thickness 0.25 μm Flow rate Constant pressure (10.3 psi). Normalinitial flow rate 34 cm sec⁻¹ (2.0 ml min⁻¹) Column oven 70° C.initially then ramp 5° C. min⁻¹ to 250° C. Hold at 250° C. for 15minutes. Injector temp 250° C. Detector temp 325° C. Injection Vol 1 μl,split ratio 2.5:1 Run time 45 minutes Fuel gases Hydrogen 40 ml min⁻¹Air 450 ml min⁻¹ Helium 45 ml min⁻¹

b) Standard Preparation

Stock standard solutions of CBD, CBN and Δ⁹ THC in methanol atapproximately 1 mg ml⁻¹ are stored at −20° C. Diluted working standards(0.1 mg/ml for Δ⁹ THC and CBD and 0.01 mg/ml for CBN) are prepared inmethanol from the stock standards and stored at −20° C. (maximum periodof twelve months after initial preparation). Allow an aliquot pipettedinto an autosampler vial to equilibriate to room temperature prior touse in a GC assay.

c) Sample Preparation

Samples of final products, i.e. “pure” cannabidiol, are diluted inmethanol prior to HPLC analysis. Optimal dilutions may be determinedempirically.

Cannabis plant material samples are prepared by taking 100 mg choppeddried material and treating this with 5 or 10 ml of Methanol/Chloroform(9:1 v/v). Extract the sample in an ultrasonic bath for 15 minutes andallow to stand in the dark for 18 hours.

d) Chromatography Procedure

Standard solutions are used to provide quantitative and retention timedata. These can be typically injected in triplicate prior to theinjection of any sample solutions and then singularly at suitableintervals during the run, with a maximum of 10 test samples in betweenstandards.

TABLE 6 Retention times THCV 33.7-34.5 minutes CBD 35.6-36.3 minutes Δ⁹THC 37.2-38.1 minutes CBN 38.5-39.1 minutes

TLC Analysis

The qualitative composition of final products and starting materials mayalso be monitored by TLC.

TLC uses both retention time and characteristic spot colour toeffectively identify the cannabinoid/cannabinoid acid components in acomplex mixture. Methanolic solutions of the final products and startingmaterial, plus standards, are prepared for TLC. An aliquot is spottedonto a TLC plate, alongside suitable reference samples (e.g. for atleast Δ⁹ THC and CBD). Following exposure to Fast Blue B reagent, THCand THCA present as pink spots, while CBD and CBDA are orange in colour.Neutrals can be distinguished from the acids by comparison of the Rfvalue to that obtained for the standards. Identity is confirmed bycomparison of Rf and colour of the sample spot, to that obtained for theappropriate standard.

A typical TLC protocol is as follows:

a) Materials and Methods Equipment:

Application device capable of delivering an accurately controlled volumeof solution i.e 1 μl capillary pipette or micro litre syringe.

TLC development tank with lid

Hot air blower

Silica gel G TLC plates (SIL N-HR/UV254), 200 μm layer with fluorescentindicator on polyester support.

Dipping tank for visualisation reagent.

Mobile phase 80% petroleum ether 60:80/20% Diethyl ether. Visualisationreagent 0.1% w/v aqueous Fast Blue B salt BN (Sigma Corp) (100 mg in 100ml de-ionised water). An optional method is to scan at UV 254 and 365nm.

b) Sample Preparation i) Herbal Raw Material

Approximately 200 mg of finely ground, dried cannabis is weighed into a10 ml volumetric flask. Make up to volume using methanol:chloroform(9:1) extraction solvent.

Extract by ultrasound for 15 minutes. Decant supernatant and usedirectly for chromatography.

ii) Final Products

The final products (crystalline CBD) are dissolved in methanol to asuitable concentration (which may be determined empirically) then useddirectly for chromatography. All sample preparations should produce afinal concentration of about 0.5 mg/ml.

iii) Botanical Drug Substance

Accurately weigh approximately 50 mg of botanical drug substance into a25 ml volumetric flask. Dissolve to make volume with HPLC grademethanol.

c) Standards

0.1 mg/ml Δ⁹-THC in methanol (Sigma).

0.1 mg/ml CBD in methanol (Sigma).

The standard solutions are stored frozen at −20° C. between uses and areused for up to 12 months after initial preparation.

d) Test Solutions and Method

Apply to points separated by a minimum of 10 mm.

-   i) either 5 μl of herb extract or 1 μl of pure cannabinoid/enriched    extract solution or 1 μl of diluted column eluate as appropriate,-   ii) 5 μl of 0.1 mg/ml Δ⁹-THC in methanol standard solution,-   iii) 5 μl of 0.1 mg/ml CBD in methanol standard solution.

Dry the prepared plate with a hot air blower.

Place the base of the TLC plate in a development tank containing themobile phase and saturated with vapour.

Elute the TLC plate through a distance of 8 cm, then remove the plate.Allow solvent to evaporate from the plate and then repeat the elutionfor a second time (double development). Remove plate and allow it to dryin air.

The entire plate is briefly immersed in the Fast Blue B reagent untilthe characteristic red/orange colour of cannabinoids begins to develop.The plate is removed and allowed to dry under ambient conditions in thedark.

Cannabinoids will give an orange-purple colour:

Cannabidiol CBD orange (fastest running) Δ⁹ Tetrahydrocannabinol THCpink Cannabinol CBN purple Cannabichromene CBC pink purple CannabigerolCBG orange Δ⁹ tetrahydrocannabivarin THCV purple

The corresponding acids form streaks of the same colour as the neutralcomponent spots. The acids run at lower Rf.

We claim: 1-30. (canceled)
 31. A pharmaceutical composition comprisingcannabidiolic acid (CBDA) or cannabidiolic acid propyl variant (CBDVA).32. The composition of claim 31, wherein the CBDA or CBDV is in the formof a cannabis based medicinal extract (CBME).
 33. The composition ofclaim 32, wherein the CBME further comprises volatile oils.
 34. Thecomposition of claim 33, wherein the volatile oils comprise terpenes orcarotenes.
 35. The composition of claim 34, wherein the terpenescomprise C10 terpenes, and C15 terpenes.
 36. The composition of claim35, wherein the C10 terpenes comprise myrcenes and pinenes.
 37. Thecomposition of claim 35, wherein the C15 terpenes comprisecaryophyllene.
 38. The composition of claim 32, wherein the CBME is amethanolic or sub-critical carbon dioxide (CO₂) extract.
 39. Thecomposition of claim 38, wherein the sub-critical CO₂ extract isobtained under the following conditions: pressure 60 bar±10 bar andtemperature 10° C.±5° C.
 40. The composition of claim 39, wherein thecomposition has been further purified to remove one or more of: waxesand other non-specific lipid-soluble material, water, ethanol andcolour.
 41. The composition of claim 32, wherein the CBME comprisesgreater than 90% w/w CBDA or CBDVA relative to the other cannabinoidspresent, measured by HPLC with reference to tetrahydrocannabinol (acid)(THC(A)) and cannabinol (CBN).
 42. The composition of claim 41 for usein the treatment of nausea, vomiting, emesis, motion sickness or likeconditions.